Lentiviral vectors for expression of hepatitis b virus (HBV) antigens

a lentiviral vector and antigen technology, applied in the field of recombinant vaccine technology, can solve the problems of limited likelihood of endogenous promoter activation, insufficient transgene expression and insufficient promoter of mhc class ii promoter

Active Publication Date: 2019-05-30
THERAVECTYS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, deletion in the U3 region of the 3′ LTR of the viral promoter and enhancer sequences in self-inactivating lentiviral vectors limits the likelihood of endogenous promoter activation.
Yet, without an enhancer, the MHC class II promoter was found not to provide sufficient transgene expression in DCs, when administered intravenously.
Thus, the MHC class II promoter is not an adequate promoter for lentiviral vectors for induction of an immune response against an antigen via IV injection.
Moreover, the dectin-2 promoter is expressed poorly in most cell types and appears to contain an enhancer.
Thus, the dectin-2 promoter is not a good promoter for lentiviral vectors for safety reasons.
Based on these criteria, viral promoters, such as the CMV promoter, are not ideal because of the presence of strong enhancers.
Current FDA approved treatments have limited efficacy (Foundation HB.
The weakness of therapy is patients seldom establish immunologic control over HBV, response is rarely durable, patients can experience relapse of disease after years of treatment, HBV replication levels rebounds to pretreatment levels once the treatment stopped, more importantly, treatment failure is common in patients.
Prophylactic vaccines are ineffective at already infected HBV carriers or hepatitis patients.
So

Method used

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  • Lentiviral vectors for expression of hepatitis b virus (HBV) antigens
  • Lentiviral vectors for expression of hepatitis b virus (HBV) antigens

Examples

Experimental program
Comparison scheme
Effect test

example 1

Constructions

[0305]PCR amplification of the proviral region of the pTRIPΔU3-CMV-GFP(15) was performed using direct (5′-CTTACTAGTTGGAAGGGCTAATTCACT CCCAAC-3′; SEQ ID NO: 7) and reverse (5′-CATTCTAGAACTGCTAGAG ATTTTCCACACTG-3′; SEQ ID NO: 8) oligonucleotides encompassing respectively the SpeI and XbaI restriction sites. The resulting fragment was digested and cloned between the SpeI and XbaI sites of the pVAX-1 plasmid (Invitrogen, Lifetech) from which the MluI site have been deleted. The resulting plasmid was named pFLAP-CMV-GFP. The SV40 sequence was amplified by PCR from the pTRIPΔU3-CMV-GFP plasmid (using the 5′-TACCCCGGGCCA TGGCCTCCAAAAAAGCCTCCTCACTACTTC-3′ (SEQ ID NO: 9) and 5′-ACTCCCGGGTAATTTTTTTTATTTATGCAGAGGCCGAG GCCGCC-3′ (SEQ ID NO: 10) oligonucleotides), and cloned into the Pml1 site of the pFLAP-CMV-GFP, the resulting plasmid being then named pFLAP-CMV-GFP-SV. The CMV promoter was amplified with direct (5′-TACACGCGTGGAGTTCCGCGTTACATA ACTTACGG-3′; SEQ ID NO: 11) and revers...

example 2

l Production

[0311]R&D productions: Vectors can be produced by transient calcium-phosphate transfection of HEK 293T as previously describe (25).

[0312]Preclinical and GMP productions: The day before transfection, HEK 293T cells are seeded in culture medium on 24 units of Cell Factory 10 (CF-10, Nunc). Cells are transfected by a calcium-phosphate method as reported previously (25). 18 to 24 hours post-transfection, culture medium is changed with production medium corresponding to Dubelcco's modified Eagle's medium (DMEM / High modified, Hyclone) supplemented with 2% heat-inactivated fetal calf serum (FCS, PAA), 1% L-Glutamine (Gibco by Life technologies), 1% Penicillin-Streptomycin (Gibco by Life technologies), 1% Sodium Pyruvate (Gibco by Life technologies), BENZONASE® (pharma grade I, 100 000 U, Merck Millipore) and MgCL2 1M. Minimum 24 hours after medium renewal, supernatant of the 24 CF-10 is harvested and pooled. After a second BENZONASE® treatment, supernatant is clarified by filtr...

example 3

l Vector Titration

[0314]qPCR reactions: HEK 293T cells are seeded in 6-well plates (BD Falcon) in culture medium and incubated for 4 h at 37° C., 5% CO2 in moist atmosphere. Cells are transduced with 3 successive dilutions of lentiviral vector. 72 h post-incubation, cells are harvested and transduced HEK 293T cell pellets are produced. Total genomic DNA from transduced cell-pellets is extracted using a method based on QIAGEN QIAamp DNA mini kit handbook. Proviral quantification is performed using Taqman qPCR. The amplification is performed with the Master Mix (Fermentas Thermo Scientific), the Flap A (CCCAAGAACCCAAGGAACA; SEQ ID NO: 18) and Flap S (AGACAA GATAGAGGAAGAGCAAAAC; SEQ ID NO: 19) primers and LENTI TM probe (6FAM-AACCATTAGGAGTAGCACCCACCAAGG-BBQ; SEQ ID NO: 20). Normalization is performed with the quantification of the actin gene (same Mix, Actine A-CGGTGAGGATCTTCATGAGGTAGT-(SEQ ID NO: 21), Actine S-AACACCCCAGCCATGTACGT-(SEQ ID NO: 22) primers and HUMURA ACT TM probe-6FAM-C...

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Abstract

The invention relates to nucleic acids, including lentiviral vectors and lentiviral vector particles, encoding at least one Hepatitis B virus (HBV) envelop surface of genotypes A and/or C antigen, at least one polymerase of genotypes A and/or C antigen, at least one HBX protein of genotypes A and/or C antigen, at least one HBV consensus core of genotypes A and/or C antigen, and at least one HBV consensus core MHCI and MHCII epitopes of genotypes A and/or C antigen. The invention encompasses these lentiviral vectors and lentiviral vector particles, methods of making the vectors, and their use, including medicinal uses. The lentiviral vectors and lentiviral vector particles are for use in administering to humans to induce immune responses against the HBV antigens.

Description

TECHNICAL FIELD[0001]The present invention is in the field of recombinant vaccine technology and relates to improvements of lentiviral vectors, which can be used as therapeutic and prophylactic vaccines. The vectors provide improved induction of immune responses over other vectors.BACKGROUND[0002]Recombinant vaccines have been developed with the progress of recombinant DNA technology, allowing the modification of viral genomes to produce modified viruses. In this manner, it has been possible to introduce genetic sequences into non-pathogenic viruses, so that they encode immunogenic proteins to be expressed in target cells upon infection or transduction, in order to develop a specific immune response in their host.[0003]Such vaccines constitute a major advance in vaccine technology (Kutzler et al., Nat Rev Genet, 9(10): 776-788, 2008). In particular, they have the advantage over traditional vaccines of avoiding live (attenuated) virus and eliminating risks associated with the manufac...

Claims

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Application Information

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IPC IPC(8): A61K39/29A61P31/14
CPCA61K39/29A61P31/14A61K39/292C12N15/86A61K2039/5256A61K2039/5258C07K2319/00C12N2740/15011C12N2740/15043C12N2740/16011C12N2740/16043C12N2799/027C12N2840/203
Inventor JAMILUDDIN, MOHAMADBEJANARIU, ANASARRY, EMELINE
Owner THERAVECTYS
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